The route of cellular entry for most conventional drugs is diffusion across the biological membrane. For this reason, drugs tend to be small (MW<500) and amphipathic, containing both hydrophobic and hydrophilic functionalities. These characteristics engender molecules with water solubility, while allowing them to cross the nonpolar lipid bilayer of the cell membrane. In contrast, the drugs used in antisense and gene therapies are relatively large hydrophilic polymers and frequently highly negatively charged nucleic acids as well. Both of these physical characteristics preclude their direct diffusion across the cell membrane. For this reason, the major barrier to gene therapy and antisense therapy is the delivery of the drug to the cellular interior. This situation is in contrast to standard drug development in which the identification of the drug is the major barrier in development.
The route of entry for most macromolecules such as DNA and oligonucleotides into cells is endocytosis. Once taken into the cell, the endosome is either recycled back to the cell surface, or matures to a late endosome and finally to a lysosome. In this process, the pH gradually drops from 7 to 6 as the early endosome becomes a late endosome. In the late endosome, the pH drops from 6 to less than 5 as it matures into a lysosome. In the late endosome and lysosome, enzymes such as proteases, nucleases, and glycosylases digest hydrolyzable components in the compartment. To prevent degradation of nucleic acids and other potential drugs in the lysosome, the endosome must be disrupted, and the contents released, prior to its evolution into a lysosome. Once endocytosis occurs, the endosome becomes a late endosome within about 10 minutes [Mukherjee et al. 1997]. The late endosome contains hydrolytic enzymes such as proteases [Berg et al. 1995] and other hydrolytic enzymes. Additional digestive enzymes become present as the endosome evolves into a lysosome.
For material to escape from endosomes into the cytoplasm, the membrane of the endosome must be disrupted. Endosome rupture can occur by osmotic pressure causing the membrane to swell and burst [Zuber et al. 2001], by membrane disruptive agents that denature the membrane bilayer structure, or a combination of these forces. Methods for accomplishing endosomal release often rely upon the environment of the lysosome and/or endosome to trigger membrane rupture and release of its contents. For example, vectors may be substrates for lysosomal enzymes such as proteases. Proteolysis can result a activation of a membrane active compound which then destabilizes the bilayer. An example of enzyme-triggered membrane breakage is adenoviral coat proteins which change structure and membrane disruptive ability upon proteolytic cleavage [Skehel et al 2000].
The drop in pH as an endosome matures into a lysosome may also be utilized to trigger membrane disruption and content release. Viral infections often require the acidification of the endosomal compartment [Carrascol994]. To mimic this viral activity synthetically, many nonviral transfections agents have been designed with pH-dependent components. Agents that are weakly basic, pKa 5-7, can be reversibly protonated in the acidic environment of the endosome. Examples include chloroquine, polyethyleneimine, and histidylated poly-L-lysine. The effect of these buffering compounds is to increase the number of protons required for a drop in pH. It is postulated that the increased number of protons, and as a consequence their counterions, causes an increase in the osmotic pressure of the endosome, which leads to membrane rupture, the proton sponge effect [Zuber et al. 2001].
Another mechanism for pH-dependent membrane disruption is the use of agents whose interaction with a membrane is dependent upon its protonation, e.g. cholesterol hemisuccinate [Lai et al. 1985], viral coat peptides and their derivatives [Plank et al. 1998], and polypropylacrylic acid (PPA) [Cheung et al. 2001]. A common characteristic of these agents is that they are carboxylic acid- and hydrophobic group-containing molecules that become less charged as the pH drops. The decrease in charge renders the molecules more hydrophobic, and thus more membrane disruptive.
The most studied synthetic carboxylate-containing polymer for endosome disruption is PPA [Lackey et al. 2002]. The propyl group in each monomer of PPA constitute the hydrophobic groups of the polymer. Another synthetic carboxylate-containing polymer is polyethylacrylic acid (PEA). PEA is also membrane-active in a pH-dependent manner. However, depending on the molecular weight of the polymer, the onset of membrane activity for PEA occurs well below pH 6. By increasing the length of the hydrophobic group of PEA by one carbon, to produce PPA, the pH-dependence of the polymer shifts to less acidic conditions such that the onset of membrane activity occurs the more physiologically relevent pH 6.5. In addition to polyacrylic acids, polyamino acid polymers containing aspartate or glutamate residues also exhibit pH sensitive charge negative charge. Many naturally-occurring pH-sensitive polymers rely on these anionic residues for their pH dependence.